This program explores innate anti-microbial defense and inflammatory mechanisms involving the host's ability to deliberately produce reactive oxygen species (ROS). Circulating phagocytes generate high levels of ROS that serve as important microbicidal agents in response to infectious or inflammatory stimuli, which is attributed to NADPH oxidase activation. Patients with chronic granulomatous disease (CGD) suffer from NADPH oxidase deficiencies, resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. The current focus of this project explores cellular mechanisms regulating related Nox Family NADPH oxidases expressed in non-phagocytic cells (Nox1, Nox4, Duox1, Duox2), notably on mucosal surfaces (lung and gastrointestinal tract), the liver, kidney, thyroid and salivary glands, brain, and vascular tissues. Several of these non-phagocytic Nox enzymes serve in host defense and inflammatory processes, as they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by cytokines or by recognition of pathogen-associated molecular patterns. ROS produced by these oxidases also provide redox signals that affect gene expression patterns during responses to infection, oxygen sensing, growth factors, hormones, cytokines, cell differentiation, cellular senescence, programmed cell death (apoptosis). In 2010, we explored host innate immune responses to two model pathogens involving excess ROS production by non-phagocytic cells: 1) Pseudomonas aeruginosa, as a bacterial pathogen that elicits Duox-derived ROS in airway epithelial cells and 2) Hepatitis C virus (HCV), as an elicitor of excess Nox4-derived ROS in infected hepatocytes, which can lead to pro-fibrotic liver injury (cirrhosis) in chronically infected patients. Using human airway epithelial cell models we compared the effects of exposure to several airway pathogens (Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus) and showed only P. aeruginosa triggers Duox1-derived hydrogen peroxide release through a mechanism requiring extracellular calcium and exposure to freshly grown bacteria. By comparing host epithelial cell responses to various Pseudomonas mutants, we concluded that several microbial factors act cooperatively to elicit Duox activity: microbial surface factors (flagellin and lipopolysaccharide) enable adhesion to host cells, thereby promoting a more efficient Type-3 Secretion System-dependent Duox1 activation. These findings suggest a mechanism by which this pathogen is eliminated from healthy airways. In contrast, we showed that over-grown Pseudomonas cultures, as a model of the transformed state of Pseudomonas established in biofilms of chronically infected lungs, produce a redox-active virulence factor (pyocyanin) that can competitively inhibit Duox activity and produce intracellular ROS. We explored the effects of oxidative stress caused by chronic pyocyanin exposure of airway epithelial cells. Two-day pyocyanin exposure (8 micromolar) elicits secretion of several pro-inflammatory cytokines as well as epidermal growth factor receptor (EGFR) ligands that trigger transcription and release of the major airway mucins. These responses reproduce many features of the advanced cystic fibrosis disease phenotype with chronic Pseudomonas infection, suggesting that pyocyanin-mediated oxidative stress in the airway epithelium is a major determinant in airway inflammation, mucus hyper-secretion, and recruitment of circulating inflammatory cells. Our studies on innate oxidative responses to HCV infection are exploring causes of hepatic injury linked to increased transforming growth factor-beta (TGF-B) levels and hepatic fibrosis. Hepatocytes infected or transfected with HCV, or HCV core protein alone, showed increased ROS production along with increased Nox4 mRNA and protein levels. In contrast, hepatocytes expressing Nox4 short hairpin RNA (RNA interference) or a truncated, dominannt-negative form of Nox4 showed decreased ROS production when transfected with HCV. The promoters of both human and murine Nox4 demonstrated transcriptional regulation of Nox4 mRNA by HCV. Analysis of luciferase reporters tied to a series of Nox4 promoter fragments (0.7-2.4 kb) identified HCV-responsive regulatory regions modulating Nox4 expression;these human Nox4 promoter fragments were also responsive to TGF-B1. Furthermore, HCV core-dependent induction of Nox4 was blocked by TGF-B-neutralizing antibodies or the expression of dominant negative TGF-B receptor type II. Collectively, these findings identified HCV as a regulator of Nox4 expression through an autocrine TGF-B-dependent signaling mechanism. These data provide evidence that HCV-induced Nox4 contributes to ROS production that may be related to chronic HCV-induced liver disease. In efforts exploring other functional roles of Nox4 (or Renox), we are characterizing mice in which the Nox4 gene is deleted. Nox4-deficient mice exhibit a normal lifespan and phenotype in the unstressed state. Gene microarray studies are focused on identifying compensating alterations in other oxidant generating or scavenging systems to explore mechanisms maintaining normal redox homeostasis in Nox4-deficient mice. Nox4 is constitutively active, consistent with its proposed role as an oxygen-sensing enzyme. We are investigating the proposed role of Nox4 in oxygen sensing and hematopoiesis, as ROS are thought to provide feedback signals regulating renal erythropoietin synthesis. Future work will examine responses of Nox4-deficient mice to various stressors to assess potential roles of Nox4 in redox homeostasis and redox-based signaling during exposure to hypoxia, infection, or inflammation. Our advances in Duox reconstitution technology are being used to screen effects of putative Duox (or DuoxA) single nucleotide polymorphisms (SNPs) or mutations in altering oxidase function or cellular targeting, which may relate to altered susceptibilities to airway infectious or inflammatory disease (cystic fibrosis, asthma, bacterial or viral infection).